A human colon adenocarcinoma cell line, LS174T, established and characterized in our laboratory, develops organoid structures reminiscent of the patient's tumor histopathology when cultured on a perfused matrix. Synthetic hollow fibers simulate a vascular system through which low molecular weight nutrients and dissolved gases diffuse freely. LS174T clones are presently being isolated and characterized. Tissue-like arrays of clones harvested from hollow fibers are being compared to the parental cell line on the bases of morphology, isoenzyme patterns, and biological properties. Cell masses growing upon bundles of hollow fibers are being fixed in situ, prepared for light, scan, and transmission microscopy or pulsed with 3H-thymidine prior to autoradiography. Carcinoembryonic antigen (CEA) content of the extracapillary fluids and total cell mass glycocalyx, using ruthenium red staining, will be compared to the parental cell line. Membrane proteins from vesicles and from 3M KCL soluble extracts are being prepared from hollow fiber and monolayer cultured cells and analyzed using PAGE-gel electrophoresis. If some or all of the clones fail to produce glandular sturctures in hollow fiber culture, the presence of soluble differentiation-associated moieties will be assessed by the addition of the cell-free extracapillary fluids from parental population hollow fiber cultures to matrix and monolayer cloned cultures. The activity of these fluids will be assessed upon an LS174T cell strain adapted to serial growth in serum-free medium, multicellular spheroids, and other human colo-rectal trmor cell lines displaying varying degrees of differentiation in vitro. These studies may afford not only information on organotypic expression in vitro, which may be harnessed to effect phenotypic modulations of in vivo colonic neoplasms, but also provide insights into cellular interactions and molecular triggers of differentiation of mammalian epithelial tumor cells.